Conformational stability changes of the amino terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system produced by substituting alanine or glutamate for the active-site histidine 189: Implications for phosphorylation effects

Abstract

The amino terminal domain of enzyme I (residues 1–258 + Arg; EIN) and full length enzyme I (575 residues; EI) harboring active-site mutations (H189E, expected to have properties of phosphorylated forms, and H189A) have been produced by protein bioengineering. Differential scanning calorimetry (DSC) and temperature-induced changes in ellipticity at 222 nm for monomeric wild-type and mutant EIN proteins indicate two-state unfolding. For EIN proteins in 10 mM K-phosphate (and 100 mM KCl) at pH 7.5, ΔH ≅ 140 ± 10 (160) kcal mol⁻¹ and ΔC_p ≅ 2.7 (3.3) kcal K⁻¹ mol⁻¹. Transition temperatures (T_m) are 57 (59), 55 (58), and 53 (56) °C for wild-type, H189A, and H189E forms of EIN, respectively. The order of conformational stability for dephospho-His189, phospho-His189, and H189 substitutions of EIN at pH 7.5 is: His > Ala > Glu > His-PO₃²⁻ due to differences in conformational entropy. Although H189E mutants have decreased T_m values for overall unfolding the amino terminal domain, a small segment of structure (3 to 12%) is stabilized (T_m ∼ 66–68 °C). This possibly arises from an ion pair interaction between the γ-carboxyl of Glu189 and the ε-amino group of Lys69 in the docking region for the histidine-containing phosphocarrier protein HPr. However, the binding of HPr to wild-type and active-site mutants of EIN and EI is temperature-independent (entropically controlled) with about the same affinity constant at pH 7.5: K′_A = 3 ± 1 × 10⁵ M⁻¹ for EIN and ∼1.2 × 10⁵ M⁻¹ for EI.